Massive calcium-activated endocytosis without involvement of classical endocytic proteins

Abstract

We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4-64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP(2)) synthesis: PIP(2) substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5'-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP(2) induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP(2) when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP(2) and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief β-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A(2) inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side.

Figure 1.

Measurement of Cm and cytoplasmic free Ca changes during the activation of outward NCX1 currents in a BHK cell. The pipette solution contains 0.5 mM EGTA and 0.25 mM Ca (i.e., 0.4 µM of free Ca), 2 mM ATP, no GTP, and 3 µM Fluo 5N (K_d= 90 µM). When 2 mM Ca is applied for 6 s, the current rises to a peak of 120 pA and decays partially during the Ca application. Cm rises by ∼50% during this time. Peak fluorescence occurs at 5 s and decays toward baseline with a time constant of ∼4 s after deactivation of current. Upon perfusing the pipette tip with 0.5 mM of additional Ca, fluorescence rises to a steady “maximal” level in a nearly linear fashion over 1 min, whereas Cm approximately doubles and then begins to decline, as described in more detail in Fig. 4 (C and D).

Figure 2.

Internalization of membrane and NCX1 exchangers during Ca-promoted MEND with activation by ATP perfusion. (A) Uptake of FM 4–64 dye (8 µM) in a BHK cell subjected to a Ca transient in the absence of ATP followed by cytoplasmic perfusion of 2 mM ATP. As indicated below the Cm record, the cell was maintained in standard solutions without ATP or GTP for 3 min. Outward NCX1 current was activated with 2 mM of extracellular Ca for 10 s, and ∼2 min later, 2 mM ATP and 0.2 mM GTP were perfused into the cell. During the experiment, FM dye was applied and removed multiple times to monitor dye binding to the outer cell surface versus dye that had been internalized. After the Ca-activated exocytic response, surface fluorescence is increased 34% more than expected from the increase of Cm. Thereafter, the introduction of nucleotides causes a 65% decrease of Cm, and the “washable” fluorescence decreases by 60% with a corresponding increase of “unwashable” fluorescence. Dye that is trapped in vesicles and vacuoles forms a clear rim close to the cell surface (see inset and Video 1; calibration bar, 10 µm). (B) Internalization of NCX1–pHluorin fusion protein during MEND in a T-REx-293 cell. Using the same ATP perfusion protocol as in A, fluorescence originating from NCX1 at the cell surface was defined by rapid pH jumps from 6 to 8. Subsequent to the decline of Cm by ∼45%, the NCX1 fluorescence at the cell surface is decreased by >30%. This internalized pH-insensitive florescence corresponds to the percentage of NCX1 exchangers internalized during MEND, as internalized membrane does not enter acidified compartments.

Figure 3.

Two types of Ca-activated MEND responses in BHK cells, resulting in membrane recycling by different mechanisms. (A) Using standard cytoplasmic solution with 8 mM ATP and 0.2 mM GTP, the activation of Ca influx by NCX1 for 20 s causes an immediate exocytic response followed by a delayed MEND response that amounts to >50% of the cell surface over 2 min. Cycles of large exocytic responses followed by MEND can then be repeated multiple times in the same cell. (B) Using the same cytoplasmic solution as in A, with an additional 1 mM spermidine, large endocytic responses are initiated during Ca transients, rather than after Ca transients, as in A. Over the course of multiple Ca influx episodes, recovery from MEND becomes more pronounced, and MEND responses amounting to >50% of the cell surface can be repeated multiple times with recovery taking place over several minutes.

Figure 4.

Ca-activated MEND in five different protocols. Electrical parameters are monitored via 0.2–0.5-kHz/20-mV square wave voltage oscillations. (A) MEND with high cytoplasmic ATP (8 mM). From top to bottom, solid records give the calculated cell Cm in pF, conductance (G_m) in nS, membrane current (I_m) in pA, and access resistance (R_a) in MΩ. The dotted record is a representative Cm response of a cell when ATP is omitted from the pipette solution. (B) MEND incurred by perfusion of ATP into cells after nucleotide depletion and the introduction of a Ca transient without nucleotides, as in Fig. 2. The ATP/GTP-free period was 3 min, NCX1 current was activated for 3 s, and 20 s later the pipette was perfused with solution containing 2 mM ATP and 0.2 mM GTP. (C and D) MEND induced by perfusion of Ca-buffered pipette solutions. Cm responses were monitored during pipette perfusion of solutions buffered to free Ca concentrations of 5–200 µM. The experimental record illustrates MEND generated by perfusion of solution with 200 µM of free Ca (10 mM NTA and 0.5 mM ATP plus 0.1 mM GTP). The cytoplasmic solution contains 80 mM Li, 40 mM Cs, and no Na. As shown in the graph for experiments with five different free Ca concentrations, the half-maximal Ca concentration was 9 µM. Standard errors were <15%. (E) Ca-activated MEND in the presence of 1 mM cytoplasmic spermidine. MEND occurs within seconds during Ca influx via NCX1 and stops rather quickly when Ca influx is terminated. (F) Immediate Ca-activated MEND response in a cholesterol-enriched BHK cell without spermidine. Patch pipettes were dipped in a hot (60°C) mineral oil–cholesterol solution (150 mg cholesterol/1 ml oil with 10% ethanol) before seal formation. Seals were highly stable, and MEND occurred very rapidly upon activating Ca influx, with almost no detectable exocytic response.

Figure 5.

PIP₂ dependence of ATP-activated MEND. PIP2 mediates ATP-dependent MEND and induces MEND without involvement of G protein cycling. Composite results are expressed as percentage of peak Cm after Ca influx (n = 7–15). (A) Ca-activated MEND with high (8 mM) ATP is blocked by cytoplasmic GTPγS (0.5 mM). (B) Blockade of ATP-dependent MEND by GTPγS, as in A, is relieved by inclusion of the PLC inhibitor, U73122 (10 µM), in the pipette solution. (C) Pipette perfusion of 50 µM PIP₂ substitutes for ATP in the activation of MEND after a Ca transient. Bar graphs give normalized Cm in BHK cells before and after perfusion of 40 µM PIP₂ for 4 min. Before PIP₂ perfusion, cells were perfused with ATP/GTP-free solution for 4 min, followed by NCX1-mediated Ca influx for 6 s. (D) The ability of PIP₂ to induce MEND is unaffected by 0.5 mM GTPγS when PIP2 is introduced in Ca-free solution (3 mM EGTA). (E) PIP₂ is required for nucleotide reperfusion-induced MEND. Average Cm responses in BHK-NCX1 cells perfused with ATP/GTP-free solution for 4 min, followed by NCX1-mediated Ca influx for 6 s, and then perfusion of 2 mM ATP for 3 min. Inclusion of 0.1 mg/ml IPP5c in cytoplasmic solutions reduced ATP-activated MEND by >80%.

Figure 6.

Salient features of polyamine/Ca-activated MEND using nucleotide-free standard cytoplasmic solutions. (A) In contrast to other MEND-promoting agents, spermidine does not cause MEND after a Ca transient has occurred; it must be present in the cytoplasm during the Ca transient. (B) Ca-activated MEND with spermidine is often small at the first Ca influx episode but large and rapid at a second Ca transient. Thus, MEND undergoes long-term facilitation by a mechanism that does not involve phosphorylation. (C) The occurrence of MEND at the first Ca influx episode is more reliable using 2 mM EDA as polyamine in both intracellular and extracellular solutions (n = 6). (D) Whereas 0.5 mM GTPγS blocks ATP-dependent MEND (Fig. 5 A), EDA/Ca-activated MEND is unaffected (n = 7). Results in C and D are paired experiments from one batch of BHK cells.

Figure 7.

Cholesterol enrichment enables fast Ca-activated MEND without polyamines. All results use BHK cells with ATP-, GTP-, and polyamine-free cytoplasmic solution. Composite Cm changes from multiple experiments (n = 4–6) are plotted by normalizing Cm after an intervention (open squares with standard errors) to Cm values before the intervention (filled squares). (A) Treatment of BHK cells with 10 mM HPCD for 5 min does not affect in an evident manner NCX1 currents or membrane fusion responses evoked by outward NCX1 current. Cm decreases by <10% during HPCD treatment. (B) Treatment of BHK cells with cholesterol-loaded HPCD for 5 min causes an average increase of Cm of 12%. Thereafter, the activation of outward NCX1 current causes an average 36% fall of Cm within 5 s, often with no evident preceding membrane fusion response. (C) Calcium transients force cells into a MEND-permissive state for many minutes. Although treatment of cells with HPCD complexes does not cause endocytic responses in control cells (see B), cholesterol enrichment causes profound MEND over 2–4 min when treatment is initialized after an NCX1-mediated Ca transient.

Figure 8.

Composite results of experiments characterizing MEND in five protocols. Bar graphs represent the peak Cm occurring in an experiment, during or shortly after a Ca transient, in relation to Cm after the occurrence of MEND. Each dataset reflects five or more observations. (A) MEND occurring with the indicated cytoplasmic ATP concentrations in response to a single Ca influx episode of 12–16 s. (B) MEND occurring upon pipette perfusion of an NTA-buffered solution with 0.2 mM of free Ca. (C) MEND occurring upon pipette perfusion of 2 mM ATP and 0.2 mM GTP after the cells were opened and maintained without nucleotides for 3 min and exposed to one Ca influx episode for 2 s. (D) MEND occurring during a single Ca influx episode for 15 s in the presence of cytoplasmic spermidine (1 mM). (E) MEND occurring over two Ca influx episodes of 15-s duration, separated by 2 min, in the presence of cytoplasmic spermidine (1 mM). See Results for complete details.

Figure 9.

Activation of MEND by the extracellular application of 1 U/ml Bacillus cereus SMase in BHK fibroblasts with Na-free solutions. (A) BHK cell perfused for >2 min with Na-, ATP-, and GTP-free cytoplasmic solution. The application of 1 mM Ca from outside has no effect on Cm, whereas the application of Ca with SMase causes a 54% drop of Cm at a peak rate of 12% per second. Membrane current (I_m) shows no response, access resistance (R_a) increases by 10%, and membrane conductance increases transiently by 0.8 nS in parallel with the first derivative of Cm (−dC_m/dt). (B) Cytoplasmic application of SMases does not cause MEND. Perfusion of purified SMase into the cytoplasm of a BHK cell causes at most 12% decline of Cm at the same concentration that causes >50% decline of Cm from outside within seconds. (C) Reversal of SMase-induced MEND. SMase application as in A caused on average a 53% fall of Cm within 15 s. With 6 mM ATP in the cytoplasmic solution (left), Cm recovered toward baseline by 60% over 5 min, whereas Cm did not recover in the absence of nucleotides (right).

Figure 10.

Desipramine treatment does not block MEND, whereas BEL treatment blocks MEND but not exocytosis. (A) ATP-dependent MEND in response to a Ca transient is unchanged by the pretreatment of cells with 100 µM desipramine for 1 h in serum-free media, followed by further incubation with 100 µM desipramine for 1 h and execution of experiments with 100 µM desipramine in both cytoplasmic and extracellular solutions. (B) Similar treatment with desipramine fails to inhibit spermidine-dependent MEND in the absence of ATP. (C) Same solutions as in A. BHK-NCX1 cells, which were pretreated with 50 µM BEL for 1 h in serum-free media, show robust Ca-activated exocytosis, with Cm doubling over three Ca influx episodes while endocytic responses are potently inhibited. (D) Same solutions as in B, with BEL treatment as in C. BEL treatment does not block exocytic responses that occur in the absence of ATP and presence of cytoplasmic spermidine (1 mM). However, endocytic responses are completely suppressed.

Figure 11.

MEND occurs in cardiac myocytes without preceding exocytic responses and can be induced by spontaneous cycles of Ca release. (A) Rat cardiac myocyte with 6 mM of cytoplasmic ATP. MEND develops rapidly during activation of NCX1 and continues for several seconds after terminating Ca influx as cells continued to contract spontaneously. MEND reversal in myocytes is labile, becoming substantially slower from one MEND cycle to the next. Reversal was negligible when MEND was allowed to proceed to a final loss of >30% of the cell surface. (B–D) Different patterns of MEND responses observed in mouse cardiac myocytes. (B) Mouse myocyte with contraction blocked by 17 µM blebbistatin. Na/Ca exchange current is relatively large. MEND begins within 3 s of activating Ca influx, and MEND terminates when exchange current is deactivated. (C) Mouse myocyte without blebbistatin. Brief activation of reverse exchange current promotes spontaneous cycles of Ca release, accompanied by transient current changes that are evident in the current record. MEND begins with no exocytic response and terminates spontaneously as spontaneous contractile activity terminates. (D) Mouse myocyte without blebbistatin. Exchange current is negligibly small. The application of Ca activates spontaneous cycles of Ca release accompanied by transient current changes. MEND begins after extracellular Ca has been removed and during spontaneous contractile activity, and it terminates spontaneously with no evident relationship to the termination of spontaneous activity. The bright field record of this experiment is provided as Video 3.

Figure 12.

Ca-activated endocytosis involves three distinct processes. (1) Large Ca transients cause long-term changes of the outer plasmalemma monolayer that promote endocytosis (Hilgemann and Fine, 2011). Possible mechanisms include the generation of lipids that promote the growth of lipid domains (i.e., the coalescence of small domains to large domains) and/or the translocation of such lipids to the outer monolayer. (2) In synergy with spermidine, high cytoplasmic Ca causes inner monolayer changes that promote endocytosis. One possible mechanism is the coalescence of lipid domains by Ca- and lipid-binding proteins (e.g., annexins) (Chasserot-Golaz et al., 2005). (3) After membrane modification by Ca, the ATP-dependent synthesis of PIP₂ promotes MEND by Ca-independent mechanisms. The clustering of PIP₂ and PIP₂-binding proteins may promote transbilayer domain coupling and membrane buckling that in turn drives endocytosis.